With the development of targeted therapies in cancer, it is imperative to establish methods for analyzing multiple tumor biomarkers accurately and quantitatively in needle tumor biopsies. Accurately and efficiently capturing a specific "molecular portrait''of each patient's tumor plays a crucial role in the diagnosis and treatment cancers and are challenging the current method of peroxidase-based immunohistochemistry which is not stoichiometric and only one stain can be done on a section. Nanotechnology is becoming a fundamental driver of advances in cancer research. In this regard, bioconjugated quantum dots (QDs) are particularly attractive as an emerging new probe for tumor biomarker profiling owing to its high multiplexing and quantification capabilities. QDs possess unique advantages compared to organic dyes and fluorescent proteins, such as high brightness and photostability, simultaneous excitation of multiple fluorescence colors with a single excitation wavelength. Although QDs have become a new biological labeling entity, the "perfect ''QD bioconjugates, in which the number of biomolecules per nanoparticle and the orientation of the biomolecules are precisely controlled, are still not available and pressing in need for accurate and quantitative detection biomarker expression. The lack of orientation control can cause the loss of QD-ligand activity while the lack of stoichiometric control will bring about variation in signal quantification. The goal of the proposed research is to develop a panel of multi-color orientation controlled monovalent QD probes using hybrid gel electrophoresis. This gel system is highly capable of separating QDs because QDs cannot enter or be fractionated by poly-acrylamide gel. Although QDs can enter agarose gel due to its larger pore size than hybrid gel, QD separation in agarose gel cannot reach the same resolution as hybrid gel can. An antibody contains two antigen binding sites, monovalent antibody containing one antigen binding site is generated by partial reduction of a whole antibody using a reducing agent. Conjugation orientation control is realized through site-specific biotinylating sulfhydryls in the antibody hinge region and leave the antigen binding site intact. Conjugates with one copy of streptavidin per QD and monovalent QD will be separated through the hybrid gel and recovered using a lab-made eluter device. The standard immunohistochemistry procedures will be followed using formalin-fixed and paraffin-embedded (FFPE) prostate cancer cell lines. Hyperpectral imaging system will be applied to perform data acquisition and quantitative analysis. Novel monovalent QD probes will provide an unprecedented precision tool in determining the molecular fingerprints of individual cancers, on which accurate diagnosis and treatment decision can be made. Our results will be relevant to the mission of the NIH and be broadly interesting to researchers studying pathology of a variety of diseases. PUBLIC HEALTH RELEVANCE: This project will conduct tumor biomarker profiling by using ongoing synthesis of a panel of multi-color monovalent Quantum Dot probes, the predicting results of accurate quantitative measurement tumor markers will provide the essential information for cancer diagnosis and personalized treatment. Our research will be relevant to the mission of the NIH and be broadly interesting to researchers studying the pathology of variety diseases.